S32354

At day 14, 2D and 3D cultures were fixated in 4% (v/v) buffered formaldehyde (VWR Chemicals) and washed with PBS. Heat-mediated antigen retrieval was performed on all samples, for 15 min at 90°C in 0.5M EDTA buffer pH 8.0 (Invitrogen, ThermoFisher). Cells were permeabilized using 0.2% (v/v) Triton X-100 (Merck Millipore) in PBS for 10 min and washed with PBS. Samples were then blocked with 50 mg/mL BSA in PBS for 1 h at room temperature (RT) and subsequently washed in PBS. Immunodetection was performed by incubation with purified anti-human CD19 antibody (10 μg/mL, SJ25C1, Biolegend) or purified anti-human CD138 antibody (10 μg/mL, MI15, Biolegend) overnight at RT, followed by biotinylated sheep anti-mouse (1:200, RPN1001; GE Healthcare) for 1 h at RT, and Alexa Fluor 488 anti-streptavidin (4 μg/mL, S32354; Thermo Fisher Scientific) for 1 h at RT, all in PBS containing 50 mg/mL BSA and incubated on a plate shaker (150 rpm). All antibodies were centrifuged before use (13,000 rpm for 5 min). Between staining steps, samples were washed in 0.1% Tween 20 (v/v, Merck Millipore) in PBS. Samples were then stained for Phalloidin–Atto 700 (0.4 nmol/mL, 79,286, Sigma Aldrich) and DAPI (100 ng/mL, D9542, Sigma Aldrich), washed in PBS and mounted using Vectashield (Vector Laboratories).